Inhibition of Wnt/β‐catenin signaling upregulates Nav1.5 channels in Brugada syndrome iPSC‐derived cardiomyocytes

Abstract The voltage‐gated Nav1.5 channels mediate the fast Na+ current (I Na) in cardiomyocytes initiating action potentials and cardiac contraction. Downregulation of I Na, as occurs in Brugada syndrome (BrS), causes ventricular arrhythmias. The present study investigated whether the Wnt/β‐catenin signaling regulates Nav1.5 in human‐induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). In healthy male and female iPSC‐CMs, activation of Wnt/β‐catenin signaling by CHIR‐99021 reduced (p < 0.01) both Nav1.5 protein and SCN5A mRNA. In iPSC‐CMs from a BrS patient, both Nav1.5 protein and peak I Na were reduced compared to those in healthy iPSC‐CMs. Treatment of BrS iPSC‐CMs with Wnt‐C59, a small‐molecule Wnt inhibitor, led to a 2.1‐fold increase in Nav1.5 protein (p = 0.0005) but surprisingly did not affect SCN5A mRNA (p = 0.146). Similarly, inhibition of Wnt signaling using shRNA‐mediated β‐catenin knockdown in BrS iPSC‐CMs led to a 4.0‐fold increase in Nav1.5, which was associated with a 4.9‐fold increase in peak I Na but only a 2.1‐fold increase in SCN5A mRNA. The upregulation of Nav1.5 by β‐catenin knockdown was verified in iPSC‐CMs from a second BrS patient. This study demonstrated that Wnt/β‐catenin signaling inhibits Nav1.5 expression in both male and female human iPSC‐CMs, and inhibition of Wnt/β‐catenin signaling upregulates Nav1.5 in BrS iPSC‐CMs through both transcriptional and posttranscriptional mechanisms.

. In addition, cardiac activation of Wnt/β-catenin signaling at early embryonic stage reduced Na v 1.5 protein in adult mouse hearts (Gillers et al., 2015;Li et al., 2018). However, because of species differences between rodent and human hearts, it is not known if Wnt/β-catenin signaling regulates Na v 1.5 in human cardiomyocytes and if inhibition of Wnt/β-catenin signaling can rescue the reduced Na v 1.5 in patient cardiomyocytes.
In this study, we demonstrate that activation of Wnt/βcatenin signaling in healthy human iPSC-derived cardiomyocytes (iPSC-CMs) reduces SCN5A mRNA and Na v 1.5 protein leading to reductions in I Na density and alterations in I Na gating kinetics. Moreover, upregulation of Na v 1.5 was achieved in BrS iPSC-CMs after inhibition of the Wnt/β-catenin signaling by two different strategies: a small-molecule inhibitor and shRNA-mediated βcatenin knockdown. Importantly, βcatenin knockdown restored the peak I Na density in BrS iPSC-CMs to near (78% of) that in healthy iPSC-CMs.

| METHODS
The generation of human iPSC lines were completed in Dr. Joseph C. Wu's lab at Stanford Cardiovascular Institute with Institutional Ethics Committee approved protocols (IRB 29904 and SCRO 485) and informed written consents were given prior to the inclusion of subjects in the study. Investigations involving human cells conformed to the principles outlined in the Declaration of Helsinki and were approved by the institutional review committee at the University of Ottawa Heart Institute.

| Human-induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (iPSC-CMs)
The generation of iPSCs from two BrS patients has been described previously (Liang et al., 2016). The first patient (male, 44 years old, iPSC ID: SCVI128-C2, designated as BrS Line 1 in this study) had unstable ventricular tachycardia and ECG revealed a pattern characteristic of BrS. This patient contained a benign variant (R620H) and a disease-causing variant (R811H) in each of the two SCN5A alleles (Calloe et al., 2013;Liang et al., 2016). The second BrS patient (male, 53 years old, iPSC ID: SCVI129-C1, designated as BrS Line 2) harbored a 1-base pair deletion mutation (causing a frame shift mutation, 4190ΔA) in one of the two SCN5A alleles, which is anticipated to generate a truncated non-functional Na v 1.5 (Liang et al., 2016). The healthy human iPSC line (ID: SCVI273, designated as Healthy Line 2) was generated in Dr. J.C. Wu's lab from cardiac I Na is found in pigs after myocardial infarction (Pu & Boyden, 1997), in heart failure patients (Shang et al., 2007;Valdivia et al., 2005), and in dogs with heart failure (Maltsev et al., 2002). Reduced I Na causes slow conduction (Park et al., 2015) and induces lethal ventricular arrhythmias (Papadatos et al., 2002;Park et al., 2015).
Reduced I Na is also responsible for the most common type of Brugada syndrome (BrS, Type 1) (Brugada & Brugada, 1992;Kapplinger et al., 2010) and a portion of the early repolarization (ER) syndrome (Antzelevitch & Yan, 2015;Watanabe et al., 2011), both of which are associated with malignant ventricular arrhythmias and sudden deaths but no disease-specific therapies are available. BrS and ER syndromes are two forms of the J-wave syndrome (Antzelevitch & Yan, 2015) and differ in the magnitude and location of J-waves on surface ECG. Reduced I Na exaggerates the transmural dispersion of repolarization in ventricular myocardium, manifested in ECG as J-waves (deflections immediately after the QRS complexes) (Antzelevitch & Yan, 2010) and triggers ventricular tachyarrhythmias. Type 1 BrS is caused by mutations in SCN5A gene (encoding the pore-forming α subunit of cardiac I Na , Na v 1.5) leading to I Na reductions via various mechanisms (Kapplinger et al., 2010;Meregalli et al., 2009;Tan et al., 2003;Vatta et al., 2002;Wang et al., 2000). Recent studies have also found a lower expression level of SCN5A/Na v 1.5 in BrS cardiomyocytes (Bersell et al., 2022;Gaborit et al., 2009;Liang et al., 2016), suggesting that reduced Na v 1.5 channel expression may be an additional pathogenic mechanism. Previous studies have shown that slowing Na v 1.5 channel inactivation with dimethyl lithospermate B (extracted from Chinese herbs) increased I Na and attenuated arrhythmias in a pharmacologically induced Brugada model (Fish et al., 2006). However, strategies to increase Na v 1.5 level for correcting the reduced I Na in BrS are lacking.
The Wnt signaling is evolutionally conserved and is a critical regulator of gene expression (Cadigan & Nusse, 1997;Liang et al., 2020). In the canonical Wnt pathway ( Figure 1A), Wnt receptor activation leads to inhibition of GSK-3β, a key mediator of βcatenin degradation; this causes cytosolic accumulation of βcatenin, which then translocates into the nucleus for regulation of target gene transcription. Increased activity of the Wnt/β-catenin pathway has been found in arrhythmogenic heart disease, such as myocardial infarction and heart failure (Dawson et al., 2013;Hou et al., 2016;Malekar et al., 2010). We and others have previously demonstrated that Wnt/β-catenin signaling reduces Na + channel transcript (Scn5a), protein (Na v 1.5), and current (I Na ) in neonatal rat ventricular myocytes (NRVMs) (Liang et al., 2015;Lu et al., 2020) and in HL-1 cells Zhao et al., 2019). Consistent with a selective effect on I Na , the L-type Ca 2+ current was not affected by Wnt/β-catenin signaling in NRVMs a healthy volunteer (female, 41 years old) as described (Kitani et al., 2019).

Wnt/β-catenin signaling in iPSC-CMs
To activate Wnt signaling, iPSC-CMs were treated with 5 μM CHIR-99021 (Selleck Chemicals, S1263, stock solution was made with DMSO at 10 or 100 mM) for 48 h before RNA extraction, qRT-PCR, and patch-clamp studies as described below. Control cells were cultured in medium containing equal volume of DMSO (Sigma-Aldrich, D2650). To suppress Wnt signaling, BrS iPSC-CMs were transduced with adenovirus expressing an shRNA targeting human CTNNB1 encoding βcatenin (Ad-shRNA-βcatenin, Vector Biolabs, shADV-206246) or control adenovirus expressing a non-silencing shRNA (Ad-shRNA-control, Vector Biolabs, 1781) at a MOI of 10. At Day 5-8 after virus transduction, cells were used for analyses (qPCR, western blot, and patch-clamp). In another group of studies, Wnt signaling was inhibited in BrS iPSC-CMs by including Wnt-C59 (4 μM, Selleck Chemicals, S7037) in culture medium for 3 days. Control cells were cultured in medium containing equal volume of DMSO (Sigma-Aldrich, D2650).

| Real-Time quantitative PCR
Total RNA was isolated from iPSC-CMs with a RNeasy mini kit (Qiagen, 74104) and cDNA was synthesized with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Real-time quantitative PCR was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad) using iTaq Universal SYBR Green Supermix (Bio-Rad, 1725121). Information on the qPCR primers was included in Table 1

| Electrophysiology
Electrophysiology experiments were carried out using standard whole-cell patch-clamp technique (Liang et al., 2014;Liang et al., 2015) with an AxoPatch 200B amplifier (Molecular Devices) at a sampling rate of 20 kHz and low-pass Bessel-filtered at 5 kHz. Pipettes were pulled from borosilicate glass tubes (OD:1.50 mm, ID: 1.17 mm, with filament, Warner Instruments, G150TF-3) using a temperature-controlled pipette puller (HEKA, PIP6). Pipettes had tip resistances of 2-5 MΩ when filled with an internal pipette solution containing (in mM): NaCl 5, CsF 125, EGTA 10, HEPES 10, and Mg-ATP 5 (pH = 7.2 with CsOH). iPSC-CMs were placed in a perfusion chamber (Warner Instruments, RC-22) on the stage of an inverted microscope (Olympus, IX-50). Voltage-gated Na + currents were recorded in voltage-clamp mode at room temperature with cells perfused in a bath solution containing (in mM) NaCl 20, TEA-Cl 50, CsCl 67, MgCl 2 1, CaCl 2 1, glucose 10, and HEPES 10 (pH = 7.4 with CsOH) (E Na = +35.3 mV). In some studies, NaCl in the bath solution was increased to 100 mM (E Na = +75.7 mV) with reductions in TEA-Cl and CsCl to keep the osmolarity constant. Either 0.1 mM CdCl 2 or 10 μM nifedipine was included in the bath solution to inhibit L-type Ca 2+ current during the whole-cell recording. Series resistance was compensated by 70%-80%. Cells were held at −120 mV and I Na was elicited by a family of 50-ms (or 200-ms) voltage steps to potentials ranging from −90 to +30 mV (when 20 mM Na + bath solution was used) or from −90 to +70 mV with 5-mV increments and a cycle length of 600 ms. In some recordings, cells were held at −90 mV and stepped to −120 mV for 200 ms before the voltages steps to elicit I Na (Zhang et al., 2018). I Na conductance and gating kinetics were measured using protocols described in figure legends.

| Statistical analysis
Data are expressed as mean ± standard deviation (SD) with p < 0.05 considered significant. Information on sample numbers, including the number of cells or samples (n) and the number of cell differentiations (N), is included in the figure legends. Differences between two means were evaluated by two-tailed Student's t-test. Differences among multiple means were assessed by one-way analysis of variance (ANOVA). When significance was detected by ANOVA, differences among individual means were evaluated post hoc by Bonferroni's test.

| Wnt/β-catenin signaling reduces
SCN5A mRNA, Na v 1.5 protein and I Na in male iPSC-derived cardiomyocytes Activation of Wnt/β-catenin signaling was induced in male healthy iPSC-CMs (Healthy Line 1) by treatment with CHIR-99021 (CHIR) for 48 h. CHIR is a cell-permeant small-molecule inhibitor of GSK-3β and is a commonly used Wnt/β-catenin pathway activator ( Figure 1A). The increases in AXIN2 mRNA ( Figure 1B), a known target of the Wnt/βcatenin pathway (Jho et al., 2002;Liang et al., 2015), suggests successful activation of the pathway by CHIR treatment. In the same CHIR-treated samples, the transcript of SCN5A, encoding Na v 1.5, was reduced ( Figure 1B). Results from three different experiments were summarized in Figure 1B. The smaller changes in AXIN2 and SCN5A mRNA in experiment #1 are likely due to the reduced activity of the CHIR molecules after prolonged storage (>6 months) in stock solution. Therefore, CHIR stock solutions that were prepared within 3 months were used in subsequent studies to reduce variations among the experiments. Regardless, the changes in SCN5A and AXIN2 mRNA were highly correlated (p = 0.0021, Figure 1B). This suggests that the reductions in SCN5A mRNA in CHIR-treated cells are a direct effect of Wnt/β-catenin signaling activation.
Western blot revealed two distinct bands for Na v 1.5 in iPSC-CMs with one band near 260 kDa and another band with a higher molecular weight ( Figure 1C, arrowhead). CHIR treatment for 48 h led to a 93% reduction (p < 0.0001) in higher band and a 63% reduction (p = 0.0035) in lower band ( Figure 1C), as well as a 77% reduction in the higher-to-lower band ratio (p < 0.001), and an 80% reduction in total Na v 1.5 (p = 0.0002). To investigate if reduced Na v 1.5 protein is associated with changes in voltage-gated Na + current (I Na ), whole-cell patch-clamp recording was performed in two different ionic conditions in male healthy iPSC-CMs. In the first experiment, I Na was recorded with a bath solution (Liang et al., 2015) containing 20 mM Na + (E Na = +35 mV) and 0.1 mM Cd 2+ (to inhibit L-type Ca 2+ current in wholecell recording). CHIR treatment led to an 82% reduction (p < 0.05) in peak I Na density (−10.5 ± 8.3 pA/pF at −20 mV, n = 9 cells, vs. control −58.0 ± 53.14 pA/pF, n = 6 cells, Figure 1D). In the second experiment, the bath solution contained 100 mM Na + (E Na = +76 mV) without Cd 2+ (instead, nifedipine was included to block L-type Ca 2+ current). CHIR treatment led to an 84% reduction in peak I Na density (−22.3 ± 7.9 pA/pF at −20 mV, n = 7 cells vs. control −138.3 ± 20.0 pA/pF, n = 12 cells, Figure 1E). Accordingly, I Na conductance was reduced by 76% after CHIR treatment ( Figure 1Ec). In both experiments ( Figure 1D,E), the apparent reversal potentials of the currents were consistent with the calculated equilibrium potentials of Na + (E Na ), suggesting that the recorded current was primarily carried by Na + .
The reduced I Na density in CHIR-treated iPSC-CMs is consistent with our previous study (Liang et al., 2015) showing reduced I Na density in rat cardiomyocytes after CHIR or Wnt3a treatment. However, in contrast to observations in rat cardiomyocytes in which I Na gating kinetics were not affected (Liang et al., 2015), I Na gating kinetics were altered by CHIR treatment in iPSC-CMs: the half-maximal activation voltage (V 1/2 ) was increased by 13.9 mV (−20.1 ± 0.4 mV in CHIR group, n = 7 cells vs. −34.0 ± 0.3 mV in control, n = 8 cells, Figure 1Ed) and k values (slope factor) were also increased (7.5 ± 0.3 vs. control 4.8 ± 0.1, p < 0.01). In addition, the steadystate inactivation curve of I Na showed a small (4.8 mV) shift to the left (V 1/2 = − 81.2 ± 0.4 mV, n = 5 cells vs. control −76.4 ± 0.2 mV, n = 14 cells, p < 0.01, Figure 1Ee). Recovery of I Na from inactivation was delayed (p < 0.05) by CHIR (time for 50% recovery, t = 16.6 ± 1.6 ms, n = 5 cells, vs. control 8.7 ± 0.3 ms, n = 7 cells, Figure 1Ef). These CHIR-induced alterations in I Na kinetics will reduce the I Na amplitude during an action potential, which is known to be proarrhythmic in the heart.

protein in female iPSC-derived cardiomyocytes
To investigate if the inhibition of Na v 1.5 by Wnt/ βcatenin signaling is reproduced in a different iPSC line and if this effect is dependent on the sex of the cells, the effects of CHIR were further studied in iPSC-CMs derived from a healthy female volunteer. CHIR treatment of female iPSC-CMs induced a 71% reduction in SCN5A mRNA (0.29 ± 0.08, n = 8, vs. control 1.0 ± 0.15, n = 9, Figure 2a) and a 69% reduction in the higher Na v 1.5 band (0.31 ± 0.17, n = 5, vs. control 1.0 ± 0.31, n = 6, Figure 2b,c). However, the lower Na v 1.5 band was not affected (p = 0.488) by CHIR treatment in female iPSC-CMs. Regardless, total Na v 1.5 was consistently reduced by CHIR in both male and female iPSC-CMs. The higher and lower bands for Na v 1.5 were further investigated by probing the same western blot samples of female iPSC-CMs with two different anti-Na v 1.5 antibodies: a monoclonal antibody that binds to the C-terminus of the Na v 1.5 protein, and a polyclonal antibody that binds to the intracellular loop between domains I and II (Figure 2d, left panel). As shown in Figure 2d (right panel), only the higher band was detected by both antibodies. We have previously identified two mechanisms that underlie the inhibition of Scn5a/Na v 1.5 by Wnt/β-catenin signaling in rat cardiomyocytes (Lu et al., 2020). The first mechanism is direct repression of Scn5a gene expression by the binding of TCF4 (the downstream effector of βcatenin) to Scn5a promoter via two sites that are highly conserved among species including human and rats (Lu et al., 2020). The second mechanism is Wnt signaling-induced upregulation of Tbx3, a potent repressor of Scn5a/Na v 1.5/I Na (Lu et al., 2020). In female healthy iPSC-CMs, CHIR treatment increased in both TBX3 mRNA and protein (Figure 2e,f), consistent with our previous observations in rat cardiomyocytes (Lu et al., 2020), suggesting that the upregulation of Tbx3 by Wnt/β-catenin signaling is conserved in both rat and human cardiomyocytes. F I G U R E 1 Wnt/β-catenin signaling reduces SCN5A mRNA, Na v 1.5 protein, and I Na in male iPSC-derived cardiomyocytes. (A) Diagram of the Wnt/β-catenin pathway which can be activated by CHIR that inhibits GSK-3β, a key mediator of βcatenin (β-cat) degradation; βcatenin translocates into nucleus and, together with TCF, regulates the transcription of target genes. GSK-3β, glycogen synthase kinase 3β; TCF, T-cell factor. (B) qRT-PCR showing correlation between reductions in SCN5A mRNA and increases in AXIN2 mRNA in male healthy iPSC-CMs after treatment with CHIR for 48 h (5 μM, n = 8 samples from three differentiations). Data were normalized to values in vehicle (DMSO)-treated cells (n = 12 samples from three differentiations). The smaller changes in AXIN2 and SCN5A mRNA in experiment #1 are likely due to the reduced activity of the CHIR molecules after prolonged storage (>6 months) in stock solution. Data were analyzed by two-tailed t-test. (C) Left panel: representative western blot showing reduced Na v 1.5 (using a polyclonal antibody) in male iPSC-CMs after treatment with CHIR for 48 h. Calnexin was used as a loading control. Right panels show quantification of Na v 1.5 higher band, lower band, the higher-to-lower band ratio, and total Na v 1.5 (both bands). For both groups: n = 5 samples from two differentiations. Data were analyzed by two-tailed t-test. (D) I Na recorded from male iPSC-CMs with a bath solution containing 20 mM Na + (E Na = +35 mV) and 0.1 mM Cd 2+ (to inhibit L-type Ca 2+ current). (a) Voltage protocol used to elicit I Na . Cells were held at −120 mV and I Na was elicited by 50-ms voltage steps ranging from −90 to +30 mV with 5-mV increments. (c) I Na conductancevoltage relationship derived from data shown in panel Eb. Conductance was calculated by dividing the current density by the driving force (E m -Reversal Potential) for each testing voltage (E m ). The two curves (black for control, n = 8 cells and blue for CHIR, n = 7 cells) were fitted with Boltzmann equation: y = A2 + (A1-A2)/(1 + exp((x-x0)/dx)) where A2 is the conductance in the unit of nano Siemens per pico Farad (nS/pF). (d) Steady-state activation curves. Colored curves (black for control and blue for CHIR) were fitted with Boltzmann equation: as shown above where x0 is the E m for half-maximal activation of I Na (voltage for 50% activation, V 1/2 ), and dx is the k value (slope factor). V 1/2 were −34.0 ± 0.3 mV in control (n = 8 cells) and − 20.1 ± 0.4 mV in CHIR group (n = 7 cells). k values were 4.8 ± 0.1 in control and 7.5 ± 0.3 in CHIR group (p < 0.01). (e) I Na steady-state inactivation curves. Cells were held for 1 second at different potentials from −120 to −45 mV before a step to 0 mV to elicit I Na . Voltages for 50% inactivation (V 1/2 ) were − 76.4 ± 0.2 mV for control (n = 14 cells) and − 81.2 ± 0.4 mV for CHIR (n = 5 cells, p < 0.01). k values were 6.3 ± 0.1 in control and 7.1 ± 0.3 in CHIR group (p < 0.05). (f) Recovery from inactivation. Two 20-ms pulses to −20 mV were applied with an interpulse potential of −120 mV at different intervals with 2-ms increments. Time for 50% recovery, t = 16.6 ± 1.6 ms for CHIR n = 5 cells versus 8.7 ± 0.3 ms for control n = 7 cells, p < 0.05.

| A small-molecule inhibitor of Wnt signaling upregulates Na v 1.5 protein in Brugada syndrome iPSC-CMs
The inhibition of Na v 1.5 channels by Wnt/β-catenin signaling in both human and rat (Liang et al., 2015) cardiomyocytes suggests that it is a conserved mechanism.
To explore the translational potential of these findings, we used BrS cardiomyocytes as an example to investigate if inhibition of Wnt/β-catenin signaling increases Na v 1.5 in human arrhythmogenic heart disease. We first used iPSC-CMs derived from a BrS patient (BrS Line 1) who had unstable ventricular tachycardia and two missense variants in the SCN5A gene (R620H and R811H on each of the two alleles) were identified in this patient (Liang et al., 2016). Heterologous expression studies (Calloe et al., 2013) showed that the R620H variant is benign, but the R811H variant reduced I Na by ~50%, although it was not clear if the reduction in current is caused by altered channel properties or by reduced Na v 1.5 expression. The study by Liang et al. (2016) also demonstrated that these BrS iPSC-CMs had reduced I Na as compared to healthy iPSC-CMs. In agreement with these previous findings, the present study showed that these BrS iPSC-CMs had a lower Na v 1.5 level than healthy iPSC-CMs (Figure 3a). CHIR treatment of BrS iPSC-CMs further reduced Na v 1.5 by 60% (0.40 ± 0.12, n = 9, vs. control 1.00 ± 0.12, n = 8, p < 0.0001, Figure 3a,b). This suggests that the inhibition of Na v 1.5 by Wnt/β-catenin signaling is found in both healthy and BrS cardiomyocytes. In addition, CHIR treatment also upregulated Tbx3 protein in BrS iPSC-CMs (Figure 3b), which is consistent with observations in healthy iPSC-CMs.
Considering the role of Wnt/β-catenin signaling in cardiac differentiation , we investigated if Wnt-C59 altered the maturation of BrS iPSC-CMs, which may explain the observed upregulation of Na v 1.5 in these cells. It has been demonstrated that the protein level of αsarcomeric actinin (α-SA, αactinin 2) is associated with the maturation degree of iPSC-CMs (Cai et al., 2019). In addition, upregulation of KCNJ2 mRNA, encoding K ir 2.1 and I K1 , has been demonstrated in more mature iPSC-CMs (Feyen et al., 2020). In the present study, none of these maturation-associated factors (α-SA protein, KCNJ2 mRNA or K ir 2.1 protein) was affected by Wnt-C59 treatment (Figure 3c,d), suggesting that Wnt-C59 treatment did not significantly impact the maturation of BrS iPSC-CMs.

Na v 1.5 protein in Brugada syndrome iPSC-CMs
To further investigate the regulation of Na v 1.5 by Wnt signaling inhibition, we used a different strategy to block Wnt/β-catenin signaling in BrS iPSC-CMs: βcatenin, the intracellular mediator of the signaling pathway, was knocked down by expressing an shRNA targeting the CTNNB1 gene (encoding βcatenin). Successful knockdown was confirmed by an 84% reduction in βcatenin mRNA (0.16 ± 0.16 n = 6 vs. a non-silencing control shRNA 1.0 ± 0.09 n = 7, Figure 4A) and a 74% reduction in βcatenin protein (0.26 ± 0.19 n = 15 vs. control shRNA 1.00 ± 0.10 n = 14, Figure 4Cb). Accordingly, the transcripts of AXIN2 and LEF1, two target genes of Wnt/β-catenin signaling, were reduced by 46% and 56%, respectively ( Figure 4A). As compared with Wnt-C59 (Figure 3c), βcatenin knockdown induced greater F I G U R E 2 Wnt/β-catenin signaling reduces SCN5A mRNA and Na v 1.5 protein in female iPSC-derived cardiomyocytes. (a) qRT-PCR showing reduced SCN5A mRNA in female iPSC-CMs after treatment with CHIR for 48 h (5 μM, n = 8 samples from two cell differentiations). Control group was maintained in culture medium containing equal amount of DMSO (n = 9 samples from two cell differentiations). Data were analyzed by two-tailed t-test. (b) Representative western blot showing reduced Na v 1.5 (using a polyclonal antibody) in female iPSC-CMs after treatment with CHIR or DMSO (control) for 48 h. Two Na v 1.5 bands were observed with one near 260 kDa and another with a higher molecular weight (arrowhead). (c) Quantification of Na v 1.5 band densities in panel B (normalized to calnexin), showing reduced Na v 1.5 higher band in CHIR group (n = 5 samples from two cell differentiations) as compared to control, DMSO-treated group (n = 6 samples from two cell differentiations). Data were analyzed by two-tailed t-test. reductions in AXIN2 and LEF1 mRNA, suggesting a higher degree of Wnt signaling inhibition. shRNAmediated knockdown of βcatenin increased SCN5A mRNA by 2.1-fold (2.11 ± 0.52, n = 8 vs. control shRNA 1.01 ± 0.18, n = 9, p < 0.0001, Figure 4B), but the L-type Ca 2+ channel gene CACNA1C was not affected (p = 0.445, Figure 4B).
Western blot analyses of BrS iPSC-CMs from six different batches of iPSC differentiation consistently showed Na v 1.5 upregulation after βcatenin knockdown ( Figure 4C). On average, Na v 1.5 protein was increased by 4.0-fold (4.04 ± 1.63, n = 14, vs. control shRNA 1.01 ± 0.34, n = 13, p < 0.0001 Figure 4Cc). The protein levels of α-SA and Ca v 1.2 (the α subunit of Ltype Ca 2+ channel) showed small increases only in one batch of the cells (Batch-2) after βcatenin knockdown (Figure 4Cd), suggesting possible changes in maturation of the Batch-2 cells. However, the upregulation of Na v 1.5 was observed in all the six batches of cells tested. These observations suggest that the upregulation of Na v 1.5 after βcatenin knockdown in BrS iPSC-CMs is a direct effect of Wnt/β-catenin signaling inhibition, rather than an indirect effect secondary to alterations in cell maturation.

| βcatenin knockdown increases cell surface Na v 1.5 and I Na in Brugada syndrome iPSC-CMs
Consistent with western blot data, immunocytostaining also demonstrated a greater level of Na v 1.5 in α-SApositive cardiomyocytes in BrS iPSC-CMs after βcatenin knockdown ( Figure 5A, middle panel). Co-staining with N-cadherin, a plasma membrane protein, demonstrated increased Na v 1.5 on the plasma membrane of BrS iPSC-CMs after βcatenin knockdown ( Figure 5A, bottom panel). The α-SA protein is located at the Z-discs of myofibrils in cardiomyocytes and the α-SA staining indicated organized sarcomeres in BrS iPSC-CMs of the control shRNA group suggesting a high level of maturation in these cells. Consistent with western blot data showing no changes in total α-SA protein after βcatenin knockdown, the organized α-SA expression was present in BrS iPSC-CMs of both control shRNA and βcatenin shRNA groups ( Figure 5A, middle panel). These observations provide further evidence that the maturation of the cells was not significantly affected by βcatenin knockdown.
I Na was recorded in BrS iPSC-CMs with the whole-cell patch-clamp technique using a bath solution containing 100 mM Na + (E Na = +76 mV). I Na was initially recorded in the presence of 0.1 mM Cd 2+ (to block L-type Ca 2+ current) and I Na was increased (p < 0.01) by 13-fold in the cells after βcatenin knockdown (−56.2 ± 33.7 pA/pF at −10 mV n = 9 cells vs. control-shRNA −4.3 ± 4.6 pA/ pF n = 8 cells, Figure 5B). To eliminate the I Na -blocking effects of Cd 2+ , I Na recording was repeated in the absence of Cd 2+ (nifedipine was included to block L-type Ca 2+ current), and I Na was increased (p < 0.01) by 4.9-fold in the cells after βcatenin knockdown (−107.4 ± 112.5 pA/ pF at −20 mV n = 14 cells vs. control-shRNA −21.8 ± 20.9 pA/pF n = 9 cells, Figure 5C). The smaller I Na when Cd 2+ -containing solution was used (Figure 5Bb vs. 5Cb) suggests that I Na was partly inhibited by Cd 2+ , consistent with previous reports showing Cd 2+ inhibition of cardiac I Na with an IC 50 of 0.18 mM (DiFrancesco et al., 1985). The reversal potential (E rev ) of I Na in control-shRNA group (+70 mV) was close to E Na (+76 mV) when Cd 2+ was omitted (Figure 5Cb). However, the E rev in control-shRNA group was shifted to +30 mV when Cd 2+ was present (Figure 5Bb) suggesting possible contamination of other currents in the whole-cell recording when I Na was inhibited by Cd 2+ . F I G U R E 3 Regulation of Na v 1.5 protein by Wnt/β-catenin signaling in Brugada syndrome (BrS) iPSC-CMs. (a) Representative western blot showing reduced Na v 1.5 (polyclonal antibody) in BrS iPSC-CMs after treatment with CHIR (right three lanes) for 48 h, as compared to DMSO-treated cells (control, middle three lanes). Two samples from healthy iPSC-CMs (female) were included in the first two lanes for side-to-side comparison which showed a lower level of Na v 1.5 in BrS iPSC-CMs. This Brugada syndrome (BrS) patient had a benign variant (R620H) and a disease-causing variant (R811H) on each of the two SCN5A alleles. (b) Left, western blot showing upregulation of Tbx3 and downregulation of Na v 1.5 (monoclonal antibody) in BrS iPSC-CMs after CHIR treatment. Right, quantification of Na v 1.5 band densities (normalized to calnexin) showing reduced Na v 1.5 in BrS iPSC-CMs after treatment with CHIR (n = 9 samples from three cell differentiations) as compared to control, DMSO-treated cells (n = 8 samples from three cell differentiations). Data were analyzed by twotailed t-test. (c) qRT-PCR showing reduced AXIN2 and LEF1 mRNA, two known targets of Wnt/β-catenin signaling, in BrS iPSC-CMs after treatment with Wnt-C59, a small-molecule inhibitor of Wnt/β-catenin signaling. SCN5A and KCNJ2 mRNA were not affected by Wnt-C59 treatment. n = 8 samples from two different differentiations for both control (DMSO) and CHIR groups. Data were analyzed by two-tailed t-test. (d) Western blot showing upregulation of Na v 1.5 (monoclonal antibody) in BrS iPSC-CMs after Wnt-C59 treatment (n = 8 samples from two differentiations of cells) as compared to control, DMSO-treated cells (n = 7 samples from two differentiations). Myocyte maturity markers, αsarcomeric actinin (α-SA, n = 8-9 samples from two differentiations) and K ir 2.1 (n = 4-5 samples from two differentiations), were not affected by Wnt-C59 treatment. Data were analyzed by two-tailed t-test. Compared with I Na recorded in healthy iPSC-CMs under the same ionic conditions (−138.3 pA/pF at −20 mV, shown in both Figures 5Cb and 1Eb), there is an 84% reduction of I Na in BrS iPSC-CMs (−21.8 pA/pF at −20 pA/pF, control-shRNA group in Figures 5Cb), which is consistent with a lower level of Na v 1.5 in BrS iPSC-CMs (Figure 3a). βcatenin knockdown in BrS iPSC-CMs increased I Na (−107.4 pA/pF at −20 mV) to 78% of that in healthy iPSC-CMs. This is consistent with observations in immunocytostaining that Na v 1.5 is increased in the plasma membrane of cardiomyocytes in BrS iPSC-CMs after βcatenin knockdown.

Na v 1.5 in a second line of Brugada syndrome iPSC-CMs
To investigate if the upregulation of Na v 1.5 by βcatenin knockdown is reproduced in cardiomyocytes of other BrS patients, we tested an additional BrS iPSC-CM line (BrS Line 2) that contains a mutation in one of the two SCN5A alleles (Figure 6a) as described by Liang et al. (Liang et al., 2016). The mutated allele contains a 1-base pair deletion causing a frame shift mutation (4190ΔA) in SCN5A gene, which is anticipated to generate a truncated Na v 1.5 protein starting at amino acid 1397 (located in the linker region between segments 5 and 6 of domain III, Figure 6a). Previous studies have shown that similar truncation mutations in this linker region (e.g., at amino acid 1393 or 1638) cause complete (100%) loss of channel function (Meregalli et al., 2009). Because the mutant, truncated Na v 1.5 protein lacks the C-terminal and intracellular loop between domains III and IV (Figure 6a), which are required for Na v 1.5 expression on plasma membrane (Rook et al., 2012;Ziane et al., 2010), it is unlikely that the truncated channels will interact with wild-type channels on the sarcolemma.
Our western blot studies showed that, similar to observations in BrS Line 1 (Figure 3a), BrS Line 2 iPSC-CMs also have a lower level of full-length, wild-type Na v 1.5 as compared to healthy control iPSC-CMs ( Figure 6b). However, BrS Line 2 iPSC-CMs and healthy iPSC-CMs had similar level of αsarcomeric actinin (α-SA, Figure 6b), suggesting that they have a similar level of maturation. The lower level of Na v 1.5 in BrS Line 2 iPSC-CMs is consistent with previous RNA sequencing study by Liang et al. (Liang et al., 2016) that demonstrated a lower level of SCN5A transcript in these cells as compared to healthy control iPSC-CMs. This suggests that reduced SCN5A/Na v 1.5 level contributes to the pathogenesis in this BrS patient.

| DISCUSSION
The present study demonstrated that pharmacological activation of Wnt/β-catenin signaling leads to downregulation of Na v 1.5 in both healthy and BrS human iPSC-CMs. In addition, the downregulation of Na v 1.5 is observed in both male and female iPSC-CMs suggesting that this is a conserved mechanism, consistent with the fact that the Wnt/β-catenin signaling is a fundamental signal transduction pathway in the animal kingdom (Cadigan & Nusse, 1997). The downregulation of SCN5A mRNA after Wnt/β-catenin signaling activation suggests that reduced SCN5A gene expression is the primary (if not the only) mechanism underlying Na v 1.5 F I G U R E 4 βcatenin knockdown increases SCN5A mRNA and Na v 1.5 protein in Brugada syndrome iPSC-CMs. (A) qRT-PCR showing reduced CTNNB1 mRNA (encoding βcatenin) in BrS iPSC-CMs expressing βcatenin shRNA (n = 6 samples) as compared to cells expressing a control, non-silencing shRNA (n = 7 samples). AXIN2 and LEF1 mRNA, two known targets of Wnt/β-catenin signaling, were reduced after βcatenin knockdown in BrS iPSC-CMs (n = 8 samples for both groups). Cells were from two iPSC differentiations. Data were analyzed by two-tailed t-test. (B) qRT-PCR showing increased SCN5A mRNA (n = 9 samples for control-shRNA; n = 8 samples for βcatenin-shRNA) in BrS iPSC-CMs expressing βcatenin shRNA. mRNA of CACNA1C was not affected by βcatenin shRNA. Cells were from two to three iPSC differentiations. Data were analyzed by two-tailed t-test. (C) Western blot showing reduced βcatenin (n = 15 samples) and increased Na v 1.5 (n = 14 samples) in BrS iPSC-CMs expressing βcatenin shRNA as compared to control shRNA group. Calnexin was used as a loading control. Cells from a total of six batches of iPSC differentiation were used for this Na v 1.5 western blot study (a polyclonal antibody for panels Ce and Cg, and a monoclonal antibody for panels Ca, Cd, and Cf). Panels Cd to Cg: the βcatenin shRNA groups were labeled as "+", and the control-shRNA groups were labeled as "−". Batch-1 contained three βcatenin shRNA samples with only two shown in the images. The two samples in Batch-4 were not included in the Na v 1.5 summary (panel Cc) because the control sample did not show a clear band. In panel Cg, two blots for Na v 1.5 with different exposure times are shown. Ca v 1.2 and αsarcomeric actinin (α-SA) only showed small increases in one batch of the cells (Batch-2). Panels Cb and Cc show summary data and were analyzed by two-tailed t-test.
reductions. This aligns with the well-established role of the Wnt/β-catenin signaling as a regulator of gene transcriptions (Cadigan & Nusse, 1997).
In both male and female iPSC-CMs, western blot analyses showed a higher band (above 260 kDa) and a lower band (near or slightly lower than 260 kDa) when a polyclonal anti-Na v 1.5 antibody was used. This may reflect the different levels of Na v 1.5 glycosylation as suggested by previous studies that the higher band corresponds to a fully-glycosylated, mature form, while the lower band corresponds to a partially-glycosylated, immature form (Mercier et al., 2015). The higher-to-lower band ratio was reduced by CHIR in both male and female iPSC-CMs, which suggests reduced Na v 1.5 glycosylation and may explain the alterations in I Na kinetics as observed in male iPSC-CMs. The difference in the relative abundance of the higher and lower Na v 1.5 bands in untreated cells may reflect the different channel glycosylation levels due to possible variations in cell maturation between male and female iPSC-CMs or among the different batches of cell differentiations in each iPSC-CM line. The greater reductions in total Na v 1.5 in male iPSC-CMs suggest that they are more sensitive to CHIR treatment than female iPSC-CMs. Previous studies have shown a half-life of 32-36 h for Na v 1.5 in adult dog cardiomyocytes (Maltsev et al., 2008). The apparent unchanged lower Na v 1.5 band in female iPSC-CMs at 48 h after CHIR treatment may result from the combination of accelerated channel de-glycosylation and accelerated channel degradation. An alternative explanation of the lower Na v 1.5 band is that it is a partly degraded Na v 1.5 protein missing the C-terminus, because this band was not detected when a monoclonal anti-Na v 1.5 antibody targeting the C-terminus was used. However, the validity of this alternative explanation would require the verification of the epitope specific binding of the monoclonal antibody.
This study further demonstrated that inhibition of the Wnt/β-catenin signaling, using two different strategies, upregulated Na v 1.5 in BrS iPSC-CMs suggesting that this is a potential therapeutic strategy. The increased I Na density, as measured in single cells, is consistent with immunocytostaining results showing upregulation of Na v 1.5 proteins at the plasma membrane in individual cardiomyocytes after βcatenin knockdown. This also suggests that the increased total Na v 1.5 protein in BrS iPSC-CMs after βcatenin knockdown, as demonstrated by western blot, reflects increased Na v 1.5 expression in the individual cardiomyocytes, instead of increased number of Na v 1.5-expressing cells due to altered cell proliferation or survival. Despite the clear and consistent upregulation of Na v 1.5 protein, the SCN5A mRNA was, surprisingly, either not increased or increased to a smaller degree after Wnt signaling inhibition. This suggests that, in addition to the known effect of Wnt signaling on Scn5a transcription (Lu et al., 2020;Wang et al., 2016), Wnt/β-catenin signaling may also regulate Na v 1.5 at the posttranscriptional or posttranslational level. In addition to its well-known role as the intracellular mediator of the Wnt/β-catenin pathway, βcatenin also plays a role in cell-cell junction (Valenta et al., 2012). Most of the βcatenin protein in a cell is located in the cytoplasmic side of the plasma membrane forming a complex with cadherin and other binding partners (Valenta et al., 2012). Our immunocytostaining of BrS iPSC-CMs showed that most βcatenin is co-localized with N-cadherin ( Figure 5A, top panel), a plasma membrane protein expressed in cardiomyocytes (Zuppinger et al., 2000). This plasma membrane pool of βcatenin was also reduced by βcatenin shRNA ( Figure 5A). Because direct interactions between Na v 1.5 and N-cadherin have been demonstrated in cardiomyocytes (Leo-Macias et al., 2016), it is possible that βcatenin knockdown affects the binding of Na v 1.5 to N-cadherin and other binding partners on the plasma membrane (Shy et al., 2013), leading to increased Na v 1.5 stability and a slower degradation rate. In F I G U R E 5 βcatenin knockdown increases cell surface Na v 1.5 and I Na in BrS iPSC-CMs. (A) Representative confocal images of BrS iPSC-CMs showing reduced βcatenin and increased Na v 1.5 in βcatenin shRNA group. Cells were co-stained with either αsarcomeric actinin (α-SA, a marker of cardiomyocytes) or N-Cadherin (a plasma membrane protein expressed in cardiomyocytes). The fifth column shows the expanded view of the white box in the fourth column. In the bottom panel, N-Cadherin was shown in the sixth column to show co-localization between N-Cadherin and Na v 1.5 (the fifth column). (B) I Na was recorded from BrS iPSC-CMs with a bath solution containing 100 mM Na + (E Na = +76 mV) and 0.1 mM Cd 2+ (to inhibit L-type Ca 2+ current in whole-cell recording). (a) Representative I Na recorded from BrS iPSC-CMs with adenoviral expression of a control non-silencing shRNA (left) or a shRNA targeting βcatenin (right). Dashed lines indicate zero current. (b) Current-voltage relationship of I Na in BrS iPSC-CMs expressing βcatenin shRNA (n = 9 cells from two batches of cell differentiation, red color) or control shRNA (n = 8 cells from two batches of cell differentiation, black color). *p < 0.05 versus control shRNA by two-tailed t-test. Error bars indicate standard errors of the mean for clarity. Right panel shows individual data points at −10 mV. Error bars indicate standard deviations. (C) I Na recorded from BrS iPSC-CMs with a bath solution that contained 100 mM Na + (E Na = +76 mV) without Cd 2+ (instead, nifedipine was included to inhibit L-type Ca 2+ current). (a) Representative I Na recorded from BrS iPSC-CMs expressing control shRNA (left) or βcatenin shRNA (right). Dashed lines indicate zero current. (b) Current-voltage relationship of I Na in BrS iPSC-CMs expressing βcatenin shRNA (n = 14 cells from two batches of cell differentiation, red color) or control-shRNA (n = 9 cells from two batches of cell differentiation, black color). Blue dots indicate values in healthy iPSC-CMs (copied from the control group in Figure 1Eb) for comparison. *p < 0.01 by two-tailed t-test. Error bars indicate standard errors of the mean for clarity. Right panel shows individual data points at −20 mV. Error bars indicate standard deviations. addition, calnexin, a chaperon protein in the endoplasmic reticulum that is important for protein folding and N-glycosylation (Kozlov & Gehring, 2020), appeared to be altered after Wnt signaling modulations. Future studies to quantify the changes in calnexin and their potential role in Na v 1.5 synthesis and tracking to the plasma membrane will show if this is one of the mechanisms for the regulation of Na v 1.5 by Wnt/β-catenin signaling.
Wnt/β-catenin signaling is known to regulate embryonic cardiogenesis and iPSC differentiation (Burridge et al., 2014;Gessert & Kuhl, 2010;Lian et al., 2012). It is required for the induction of cardiac mesoderm (Lindsley et al., 2006;Yamaguchi et al., 1999), but it inhibits the differentiation of cardiac mesoderm cells into cardiomyocytes (Naito et al., 2006;Ueno et al., 2007) and the maturation of early-stage cardiomyocytes (at Day 12 of iPSC differentiation) (Buikema et al., 2020). The present study investigated late-stage iPSC-CMs (at Day 30-45 of differentiation) that have matured in culture, as indicated by the organized sarcomere protein expression in the cells. The CHIR-induced reductions in SCN5A mRNA are highly correlated with the levels of Wnt signaling activation as estimated by AXIN2 mRNA increases. In addition, we and others have previously demonstrated downregulation of Scn5a/Na v 1.5 in adult mouse and rat ventricular myocardium after activation of Wnt/β-catenin signaling (Huo et al., 2019;Lu et al., 2020;Wang et al., 2021). A more recent study (after the preprint posting of the present study http://dx.doi.org/10.2139/ssrn.3815857) also showed downregulation of Na v 1.5 in healthy adult F I G U R E 6 βcatenin knockdown increases Na v 1.5 protein in a second line of Brugada syndrome iPSC-CMs. (a) Diagram of the genotype of a different line of Brugada syndrome iPSC-CMs (BrS Line 2). This BrS cell line harbors heterozygous mutation. The mutant allele contains 1-base pair deletion causing a frame shift mutation, 4190ΔA, in SCN5A gene which is anticipated to generate a truncated non-functional Na v 1.5. The monoclonal Na v 1.5 antibody binds to the C-terminus and will only detect the wild-type Na v 1.5. (b) Western blot showing similar level of αsarcomeric actinin (α-SA, a marker of cardiomyocyte maturation), but reduced level of the wild-type Na v 1.5 (polyclonal antibody) in BrS Line 2 iPSC-CMs as compared to healthy iPSC-CMs. (c) Western blot showing upregulation of the full-length, wild-type Na v 1.5 (the bands above 260 kDa) (using a monoclonal antibody) and unchanged αsarcomeric actinin (α-SA) after shRNA-mediated βcatenin knockdown in BrS Line 2 iPSC-CMs. Right panel: quantification of western blot data. n = 10 samples from two independent differentiations. Data were analyzed by two-tailed t-test. human heart slices after treatment with a GSK-3β inhibitor (SB216763) (Li et al., 2022). These observations suggest that the CHIR-induced reductions in SCN5A/Nav1.5 in iPSC-CMs are unlikely secondary to alterations in cardiomyocyte maturation. On the other hand, the upregulation of Na v 1.5 in BrS iPSC-CMs after inhibition of Wnt signaling is not accompanied by changes in maturation marker levels or sarcomere organization, suggesting that this effect is not mediated by altered cell maturation.
One limitation of the present study is that the iPSC-CMs generated using standard protocols contain different types of cardiomyocytes (i.e., atrial, ventricular and nodal myocytes) (Hamel et al., 2017), which may explain the variation in Na v 1.5 expression and I Na density among the individual cells in this study. However, our previous study (Liang et al., 2015) using pure culture of neonatal rat ventricular myocytes (NRVMs) and other studies using mouse atrial cell line Zhao et al., 2019), are consistent with findings in iPSC-CMs of this study, suggesting that the key findings can be applied to both atrial and ventricular myocytes. The second limitation of iPSC-CMs is their immature phenotype (Hamel et al., 2017;Liang et al., 2019). Although the present study used relatively mature iPSC-CMs (at 30-45 days of differentiation), future studies are needed to further investigate the effects of Wnt signaling inhibition on adult BrS cardiomyocytes before advocating it as a therapeutic strategy in the patients.
In summary, this study demonstrated downregulation of Na v 1.5 by Wnt/β-catenin signaling in both male and female iPSC-CMs with reduced SCN5A mRNA as one of the underlying mechanisms. More importantly, this study showed that blocking Wnt/β-catenin signaling is a valid strategy to restore the expression of Na v 1.5 and voltagegated Na + current in iPSC-CMs of BrS patients.

AUTHOR CONTRIBUTIONS
Conception or design of the work: A.L., D.R.D., and W.L.; Acquisition, analysis or interpretation of data for the work: A.L., R.G., C.C., Y.X., J.W., and W.L.; Drafting the work or revising it critically for important intellectual content: A.L., R.G., C.C., Y.X., J.W., D.R.D., and W.L. All authors have approved the final version of the manuscript, agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved, and all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.